Sex determination is a fundamental decision that essentially all metazoans encounter during their development. Sex determination in Drosophila melanogaster involves a hierarchy of alternative splicing decisions, and is also the best understood example of splicing regulation. Splicing is a process by which non-coding sequences (introns) are removed from the precursor messenger RNA. In higher eukaryotes, constitutive and alternative splicing are important aspects of gene regulation in many important cellular processes. Approximately 15 percent of the mutations that have been linked to human diseases affect RNA splicing signals, including cellular transformation, Duchenne muscular dystrophy, and tumor metastasis. Our goal is to understand how RNA-binding proteins recognize target RNAs and regulate constitutive and alternative pre-mRNA splicing. The Drosophila protein Sex-lethal (SXL) acts as a key binary switch between the male and female cell fates. In the past, we defined the mechanism by which SXL regulates alternative splicing by antagonizing the known splicing factor U2AF65. Specificity is an underlying theme in biological regulation. U2AF65 and SXL offer excellent models for specific RNA-protein interactions in the context of splicing regulation. For example, while the general splicing factor U2AF65 recognizes a wide variety of polypyrimidine-tract/3' splice sites, the highly specific splicing repressor SXL recognizes a specific sequence. Although both proteins contain a ribonucleoprotein-consensus motif, they have distinct RNA-binding specificity. However, it is not understood how these seemingly similar proteins achieve unique RNA-binding specificities. To define the structural basis for the RNA-binding specificities of U2AF65 and SXL, we will extend our analysis of the RNA and the proteins by using a combination of biochemical, molecular, and genetic approaches. Our findings will also be directly applicable to other members of this largest family that likely regulate different aspects of RNA biogenesis. In addition, SXL controls many female-specific functions. However, some of the relevant genes that are regulated by SXL remain to be identified. To identify these targets, we will use a combination of recently developed molecular approaches - genomic SELEX and subtractive hybridization/differential display. These approaches should complement genetic analysis.